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| SITE-DIRECTED MUTANTS, AT POSITION 166, OF RTEM-1 BETA-LACTAMASE THAT FORM A STABLE ACYL-ENZYME INTERMEDIATE WITH PENICILLIN |
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| Author(s): ADACHI H, OHTA T, MATSUZAWA H |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 266 Issue: 5 Pages: 3186-3191 Published: FEB 15 1991 |
| Times Cited: 110 References: 44 |
| Abstract: Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme. |
| Document Type: Article |
| Language: English |
| Reprint Address: ADACHI, H (reprint author), UNIV TOKYO, DEPT AGR CHEM, BUNKYO KU, TOKYO 113, JAPAN |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: EX600 |
| ISSN: 0021-9258 |
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